SPECIAL FEATURE: PERSPECTIVE

Water is an active matrix of life for cell andmolecular biologyPhilip Balla,1

Edited by Pablo G. Debenedetti, Princeton University, Princeton, NJ, and approved May 1, 2017 (received for review March 7, 2017)

Szent-Gy}orgi called water the “matrix of life” and claimed that there was no life without it. This statementis true, as far as we know, on our planet, but it is not clear whether it must hold throughout the cosmos. Toevaluate that question requires a close consideration of the many varied and subtle roles that water playsin living cells—a consideration that must be free of both an assumed essentialism that gives water analmost mystical life-giving agency and a traditional tendency to see it as a merely passive solvent. Water isa participant in the “life of the cell,” and here I describe some of the features of that active agency. Water’svalue for molecular biology comes from both the structural and dynamic characteristics of its status as acomplex, structured liquid as well as its nature as a polar, protic, and amphoteric reagent. Any discussionof water as life’s matrix must, however, begin with an acknowledgment that our understanding of it asboth a liquid and a solvent is still incomplete.

water | hydration | hydrophobic effect | protein chemistry | solvation chemistry

Liquid water is so central to life on Earth that itconditions the search for the possibility of life elsewhere.The mistaken identification of “canals” on Mars in thelate 19th century fueled speculations about life on thatplanet, including H. G. Wells’ seminal 1897 alien inva-sion fantasy TheWar of theWorlds. The possible discov-ery of recent flowing surface water on Mars (1) and theexistence of global briny oceans beneath the icy surfacesof Jupiter’s moons Europa, Callisto, and Ganymedehave kept those speculations alive. Our experience ofterrestrial life has encouraged the assumption that liquidwater is almost a sine qua non, a notion expressed in theunofficial slogan of NASA’s early astrobiological pro-gram to “follow the water.”

Despite this status, water’s roles in sustaining lifeare still imperfectly understood and have until the pastseveral decades been routinely underestimated (2).The common picture was that of a passive matrix: asolvent that simply acts as a vehicle for the diffusivemotions of functional biological macromolecules, suchas proteins and nucleic acids. It is now clear that, onthe contrary, water plays an active role in the life of thecell over many scales of time and distance (3). Al-though life on Earth seems unable to exist in any sus-tained way without it, this dependence is subtleand multifaceted.

In truth, that should not surprise us. Water is acomplex fluid in its own right (4, 5), and Darwinian

adaptation to a complex milieu might be expectedto generate much the same kind of enmeshing andinterplay of the biological and environmental at themolecular scale that we find at higher levels of life’sorganizational hierarchy. That, one might add, doesnot guarantee that water in uniquely suited to be asolvent of life (6). However, it does give aqueous lifea rich, varied, and endlessly nuanced character.

Water exhibits diverse structural and dynamicalroles in molecular cell biology (3). It conditions and infact partakes in the motions on which biomolecularinteractions depend. It is the source of one of thekey forces that dictate macromolecular conformationsand associations, namely the hydrophobic attraction. Itforms an extraordinary range of structures, most ofthem transient, that assist chemical and information-transfer processes in the cell. It acts as a reactive nucle-ophile and proton donor and acceptor, it mediateselectrostatic interactions, and it undergoes fluctuationsand abrupt phase-transition–like changes that serve bi-ological functions. Is it not rather remarkable that a sin-gle and apparently rather simple molecular substancecan accomplish all of these things? Looked at this way,there does seem to be something special about water.

These, furthermore, are just the molecular andnanoscopic roles. I will not consider, for example,hydrodynamic processes, although these might notbe insignificant even at the nanoscale (7). Control of

aPrivate address, London SE22 0PE, United KingdomAuthor contributions: P.B. wrote the paper.The author declares no conflict of interest.This article is a PNAS Direct Submission.1Email: p.ball@btinternet.com.

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water transport and osmosis, wetting properties, heat manage-ment, and other factors is important at scales from the cellularto the organismal, and indeed to entire ecosystems and habitats.Water is never far from the surface of life.

Water in the CellWhereas water in the cell was traditionally regarded as a backdrop—tobe conveniently omitted from colorful ribbon diagrams of bio-molecular structures—an alternative school of thought awardedwater a quasimystical role as an agency of life. The term “biologicalwater” sometimes denoted a putative state allegedly “tamed” andrendered biophilic by cells (8).

There is no meaningful value in the notion of biological watertoday (9). However, we also cannot assume that water in the cell isthe same as pure, bulk liquid water. Aggregate measures of waterdynamics, for example, suggest that around 10–25% of watermolecules in cells have slower reorientational dynamics, by aroundan order of magnitude, than those in the bulk (10, 11). It is generallyassumed that this “slow water” is in some way engaged in hy-drating macromolecules and other cytoplasmic solutes. However,the devil is in the details, and apportioning cell water into fractionslabeled “bound” and “free”, or “slow” and “bulk-like”, does littleto elucidate the functions that it serves.

Even the nature of bulk liquid water itself has been riven withdisputes and controversies. In many ways, these arguments stemfrom a long-standing tension between the tendency to speak of“water structure” in almost crystallographic terms and the rec-ognition that it is an inherently dynamical entity (12). Liquid waterforms a fluctuating network of hydrogen bonds, but each bondhas an average lifetime of around a picosecond. The shape of theH2O molecule encourages the formation of a tetrahedrally co-ordinated motif, which itself is the building block of ephemeralfive- and six-membered rings (13). Such ring structures create agood deal of empty space within the network, giving ice a lowerdensity than the liquid, in which defects in the network may en-able molecules to encroach into the free space. Fundamentally,water structure can be regarded as a compromise between ice-like open networks and liquid-like random close packing. Thethermodynamics of solvation in water are generally governed by abalance between the enthalpic water–water and water–soluteinteractions (hydrogen bonding, electrostatic, and van derWaals) and the entropic consequences of forming and dis-rupting relatively ordered hydrogen-bonded networks, condi-tioned by geometric factors of the interfaces and possibly themicroenvironment.

In particular, much cell water is, to some degree, confined orconstrained. The average distance between macromolecules inthe cytoplasm is around 1 nm, corresponding to just three to fourmolecular layers of water—which, simply on the basis of classicalsolvation theory, cannot be considered bulk-like. The presence ofa solute usually alters the hydrogen bond network. Some smallpolar solutes, such as urea (14), can be “fitted in” with relativelylittle solvent rearrangement, whereas small hydrophobic solutescan be enclosed in a cavity around which surrounding watermolecules preserve their hydrogen bonding by rearrangement(15). Small, simple ions are typically solvated by two or so layers ofhydration water, with no longer-ranged perturbation of bulkstructure (16). For larger surfaces, such as those of proteins,truncation of the hydrogen bond network is inevitable. At hy-drophilic interfaces, water molecules might engage in hydrogenbonding with surface groups, such as acidic residues in proteins.At hydrophobic surfaces, meanwhile, water can be considered

to form structures that preserve as much hydrogen bondingas possible.

This picture of “water ordering” around a hydrophobic particlewas the basis of the classic explanation of hydrophobic interac-tions advanced by Kauzmann (17). The attraction of hydrophobesin water is well-attested and is one of the key driving forces forprotein folding and the formation of functional multiprotein ag-gregates (18). It also sustains the self-assembly of lipid mem-branes, and matching of hydrophobic surfaces is often observedin protein–ligand binding. It is no exaggeration to say that hy-drophobic interactions are a dominant force in molecular biology.

Kauzmann’s argument (17) was that this force is entropic inorigin. If water becomes more orderly—perhaps more “ice-like”(with six-membered rings) or “clathrate-like” (with five-memberedrings)—around hydrophobic surfaces, the expulsion of that waterwhen two such surfaces come together incurs an entropic pre-mium. The picture is intuitively very appealing, which is doubtlesswhy one still sees it invoked. However, both computer simulationsand direct measurements of water structure and dynamics aroundhydrophobic solutes have failed to offer support for a more staticand orderly hydration environment (19, 20)—in some respects,quite the reverse (21). There is good reason to believe that en-tropy is indeed the main factor in the hydrophobic interaction (22),but quite how to account for this at themolecular scale—at least interms of structural pictures of hydrogen bonding—is not clear.Part of the problem here is that there is no unique way to quantifywater structure, and different choices are not necessarily consis-tent with one another (20).

In any event, there is now good reason to suppose that thehydrophobic interaction is not a single phenomenon. Lum et al.(23) proposed that there should be a cross-over between small(<1 nm) hydrophobic solutes, to which water’s hydrogen bondnetwork can adapt itself to some degree, and large hydrophobes(>1 nm), which ought instead to be considered in the context ofextended wetting and interfacial free energies (24). For smallhydrophobes, such as methane (25), the water molecules mayindeed form a clathrate-like structure around the solute, althoughnot in a static sense. Rather, the hydration shell is evidently dy-namic, although it is not clear how much so relative to the bulk:some measurements (26) have shown slower relaxation, whereasothers (27) have shown enhanced rotational mobility. The twopictures, obtained with different experimental techniques, are notnecessarily incompatible (28).

For large hydrophobes, meanwhile, Lum et al. (23) proposethat hydrophobic attraction happens via the classical phenome-non of capillary evaporation: an abrupt collective evacuation ofwater from the intervening space between surfaces at some crit-ical separation, pulling the surfaces together because of the me-nisci at the edges. This drying transition is driven by pronouncedfluctuations in water density (29), which might facilitate a non-classical nucleation mechanism for vapor cavities with a low freeenergy barrier (30).

There is reason to expect a drying transition for strongly hy-drophobic, smooth surfaces (31); whether it can occur for chemi-cally heterogeneous and nonplanar protein surfaces is less clear(32, 33). Camilloni et al. (34) have even argued, on the basis ofmodeling of NMR chemical shifts, that water molecules at theprotein surface have the same number of hydrogen bonds onaverage as those in the bulk, and therefore should be consideredto be in the “small-hydrophobe” regime in which only rotationalentropy is decreased. That extreme view, however, conflicts withevidence of dangling bonds (nonhydrogen-bonded OH groups)

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at the surfaces of proteins, especially in planar and concave re-gions (35). Moreover, enhanced water density fluctuations of thekind thought to drive dewetting (29) are observed at proteinsurfaces regardless of whether they actually undergo dewettingduring aggregation (36, 37). Although the generality of dewettingas a mechanism for the interaction between “large” hydrophobicsurfaces or patches in biology therefore remains an open ques-tion, it seems entirely plausible that evolutionary “tuning” of theproteins to bring the hydration environment close to a phasetransition of this sort offers a way to produce big effects from smallchanges in the environmental conditions (36).

That kind of delicately poised “life at the edge,” facilitatingabrupt drying of an enclosed hydrophobic space, has also beenobserved in simulations of protein channels and might account forgating of ion transport in mechano- and voltage-sensitive pores(38, 39). Here, emptying of the pore prevents ion transport notsterically but because of the excessive cost of ion dehydration inthe dry environment. Emptying of water from buried hydrophobiccavities might also create a driving force for ligand docking inthermophilic proteins (40).

In summary, hydrophobic interactions are central to the cell’ssupramolecular chemistry, but their origins are still not fully un-derstood, and in all probability there is no unique mechanism: thechemical nature, size, and geometry of the interacting particlesare all important, as are dynamical, collective fluctuations of theintervening water (41).

A word of caution is due: there is no obvious reason to thinkthat solvophobic effects, such as drying transitions, are specific towater. The question, still unresolved, is to what extent water’sidiosyncrasies guide and condition them (42). Might the enthalpicand entropic consequences of water’s ability to form relativelystructured hydrogen bond networks in small clusters and cavitiesmake it especially sensitive to the geometry of confinement, forinstance? Might water’s particularly large surface tension give it anunusually (although not extraordinarily) long evaporation lengthscale, a measure of its readiness to undergo drying transitions(43)? Or might enhanced density fluctuations be a general prop-erty of solvents in small confining geometries between sol-vophobic blocks, owing to the effects of incipient capillaryevaporation on the solvent’s local compressibility (44)? Howspecial, really, is water in its ability to mediate these macromo-lecular interactions? We might not yet be able to give a definitiveanswer to that question, but we have a better sense now of whereto search for it.

Hydration Is DynamicAs these instances already make clear, we will understand ratherlittle about water in molecular biology on the basis that biomol-ecules are surrounded by some vague sheath of hydration water.It is necessary to have a detailed, perhaps atomic-resolution pic-ture of where the water molecules are and how they fit together.This structural information has been obtained for many molecularentities—especially proteins, but also DNA and membranes—from a variety of methods, such as X-ray and neutron scattering,NMR, and second harmonic generation spectroscopies, assistedby molecular dynamics and ab initio simulations. Low-frequency,large-amplitude modes in the terahertz range are particularlyimportant in controlling the conformational changes that domi-nate protein function, and are conveniently probed using ter-ahertz spectroscopy (45, 46). However, there is no simplequalitative account of how protein and solvent dynamics interact.Fluctuations of both take place over a wide range of timescalesfrom milliseconds to picoseconds, influencing several aspects ofprotein function (Fig. 1). Because no single technique can span somany temporal orders of magnitude, there has been considerabledebate about how to reconcile the results of different experimentalmethods that explore dynamics, such as NMR relaxation, neutronscattering, ultrafast IR, and terahertz spectroscopies (28, 47).

The general picture that emerges, however, is one in whichwater molecules partake in the hydration environment with a verywide range of residence times and dynamics that may be bothfaster and slower than the bulk. In general, hydration waters at thesolvent-exposed surfaces of proteins have residence times ofseveral picoseconds, but those within deeply concave clefts andinternal cavities can be much longer-lived—up to several micro-seconds—before exchanging with the bulk (48, 49). Moleculardynamics simulations of myoglobin, for example, indicate that,although the residence times of hydration water molecules aremostly rather similar to those in bulk water (<10 ps), there is a longtail of longer residence times, with some waters in cavities andclefts—geometry, rather than surface chemistry, seems to be thedominant factor—reaching up to 450 ps (48).

Orientational relaxation too is often slowed in the hydrationshell. For example, a comparison of molecular dynamics simula-tions with femtosecond IR spectroscopy of the hydration of bovineα-lactalbumin (50) reveals a continuum of water relaxation times;slow waters have relaxation times >7 ps, and some are as much as20 ps (Fig. 2). The latter molecules again tend to be located inconcavities on the protein surface, and they make fewer hydrogenbonds with surrounding waters than do molecules in the bulk.

Fig. 1. The hierarchy of timescales for motions of proteins and their hydration environment. HB, hydrogen bond. Reproduced from ref. 46 withpermission from AIP Publishing.

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Moreover, waters near hydrophobic groups tend to be slower onaverage than those near hydrophilic groups.

A study of four diverse proteins by Fogarty and Laage (51)shows that, despite their many differences, all have rather similarhydration shell dynamics. This finding was interpreted as an in-dication that the dynamics are determined by rather generalfeatures of surface chemistry and topology, which induce ex-cluded volume effects and hinder the approach of new hydrogenbond acceptors within the hydration network.

The traditional “lock and key” picture of enzyme action haslong been modified to acknowledge the vital importance ofconformational freedom (52). In short, dynamics must collaboratewith structure to get the job done. There has been somewhatslower but now widespread recognition that the dynamical be-havior of biological macromolecules in general, and of proteins inparticular, cannot be decoupled from that of its solvent. In oneview, dynamical degrees of freedom in the hydration shell supplyfluctuations that help proteins to undergo the conformationalshifts entailed by their chemical function. For example, X-rayscattering from the collective modes of hydrated lysozyme (53)shows that a weakening of “soft” phonon modes at low hydrationis correlated with a decline in enzymatic activity. Frauenfelderet al. (54) have proposed a “unified model” of protein dynamics,in which short-wavelength fluctuations are slaved to those of thehydration layers whereas large-scale protein motions are slaved tobulk fluctuations and dominated by viscosity.

How Hydration Water Assists Protein FunctionHydration water molecules may adopt crystallographically well-defined positions around a macromolecule, and some of thesehave functional roles. One might say that the surfaces of thebiomolecules are not sharply defined: their sphere of influenceextends beyond the van der Waals surface into the solvent, andthis coupling can make the hydration shell for all intents andpurposes part of the biomolecule itself, imbued with some of theinformation that it encodes and therefore able to play a role inintramolecular rearrangements and intermolecular recognitionprocesses. Examples of how water molecules and networks at a

protein surface may assist in its recognition and catalytic functionsare legion; one might reasonably suspect that proteins treatwater as a resource to be exploited whenever convenient. Forexample, water molecules can mediate interactions between aprotein and a substrate either to increase selectivity or to enablerecognition of multiple substrates. They can transmit conforma-tional changes from one location to another; they may act aschannels for proton conduction or provide proton donor, accep-tor, and storage sites. I have outlined some instances in previousarticles (3, 55); here, I take the opportunity to indicate some re-cent ones, while pointing to some of the general strategies thatthey exemplify.

Proton Donation and Translocation. One of the most commonuses for bound water in biology is as a channel for proton trans-port, generally by means of the Grotthuss hopping mechanismthat produces anomalously fast proton transport in pure water(56). This hopping process is more complex than once thought,not least because it seems to occur over a wide range of lengthscales (57).

Hydrogen-bonded chains of water molecules may comprise“water wires” that support proton translocation into and throughproteins (58). This transport can happen in passive fashion, but itmay also be active, dynamic, and controlled by protein motions.Kaila et al. (59) describe such a process in Complex I, an enzymeinvolved in the initial step in the mitochondrial and bacterial re-spiratory process, in which proton pumping is redox driven bycoupling to electron transport between NADH and quinones.Here, a transient proton-conducting water channel is formedby the cooperative hydration of three antiporter-like subunitswithin the membrane domain of the complex. As Kaila et al. (59)conclude, “water-gated transitions may provide a generalmechanism for proton-pumping in biological energy conversionenzymes.”

Delicate marshaling of water molecules into positions thatcontrol the proton conductivity of a channel is also evident incytochrome c oxidase, a transmembrane proton pump driven byoxygen reduction. Goyal et al. (60) show how hydration seems to

Fig. 2. Water reorientational decay times seen in simulations of (Left) native and (Right) misfolded bovine α-lactalbumin. Reproduced from ref. 50with permission, copyright 2016 American Chemical Society.

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carefully tune and orchestrate this proton translocation. A gluta-mate residue is believed to act as a temporary proton donor, andits proton affinity is controlled by the degree of hydration in aninternal hydrophobic cavity. That hydration, in turn, is governedby protonation of a substituent on the heme group 10 Å away,triggering movement of a loop that gates the cavity’s entrance.

Proton transfer in the protein PsbO, a subunit of photosystem II(PSII), involves low-mobility water molecules close to the surfacethat form part of an extended water–carboxylate network (61).Some of these waters might also assist the docking of PsbO intothe PSII complex. This complex itself has a hydration shell in whichno fewer than around 1,300 water molecules can be located (Fig.3) (62). These waters seem to offer several hydrogen-bondedchannels for transporting protons and entire water molecules forphotolysis. The formation of oxygen molecules probably involvesoxidation of waters bound to the central catalytic Mn4CaO5

cluster (Fig. 3C). In one stage of the oxygen-generating cycle, ahydrogen-bonded cluster of water molecules at this site acts as acatalytic proton acceptor, storage site, and donor (63–65): anexample of bound water serving as a reactive chemical substrate.

Such exquisite orchestration of hydration water by a protein tocontrol protonation reactions might turn out to be rather com-mon. Consider, for example, the DNA repair enzyme MutY, whichexcises DNA bases damaged by hydroxyl radical oxidation. Therate-determining step of this lysis involves protonation of a ni-trogen in adenine, which is assisted by a highly organized catalyticcluster of five water molecules (66) (Fig. 4). The arrangement ofhydrophobic and hydrophilic residues around the water clusterappears to act as a “water trap” to ensure that these waters havelong residence times.

Allostery. Beyond such direct interactions of water moleculeswithin the active site, hydration networks may participate in

allosteric conformational shifts. In the hexameric multidomainprotein glutamate dehydrogenase, the opening and closing of ahydrophobic pocket are accompanied by wetting and drying ofthe pocket, whereas binding and unbinding of water molecules ina hydrophilic crevice accompany changes in its length (67). Thesetwo changes in hydration are coupled, creating a kind of “hy-draulic” mechanism for large-scale conformational change.

Hydration changes involved in allostery have been probed intime-dependent fashion by Buchli et al. (68) for the PDZ domainprotein, a common model system for allosteric processes. Byinserting an azobenzene photoswitch in the binding groove of theprotein, they could control conformational changes via photo-induced isomerization, opening this groove in much the samemanner as occurs on ligandbinding. Fast IR spectroscopy showed thata change in water density in the vicinity of the photoswitch immedi-ately after switching propagates slowly through the water networkover about 100 ns until it reaches the other side of the protein, whereit might induce a remote allosteric change in protein conformation.

Fine-Tuning of Protein Environment via Hydration Changes.

Water can help to fine-tune protein functionality in a variety ofways. It seems, for example, to enable the promiscuity of alkalinephosphatase enzymes in catalyzing the hydrolysis of a range ofdifferent phosphate and sulfate substrates. First principles simu-lations suggest that a part of the reason why this class of enzymescan support different types of transition state in the same activesite (69) is the differential placement of water molecules. Anotherform of “hydration tuning” is revealed in the chloride-pumpingtransmembrane retinal protein halorhodopsin of halophiles (70).Here, subtle rearrangements of waters and ions occur in the vi-cinity of the chromophore as chloride translocation progresses,inducing changes in chromophore bond lengths that affect itsabsorption spectrum.

Fig. 3. (A) The hydration halo of PSII. (B) The same with protein chains shown only in light gray for clarity. (C) Water molecules (W) attached to theMn–Ca complex. (D) A water channel within PSII that ferries protons to the bulk aqueous phase. Reproduced from ref. 62 with permission fromMacmillan Publishers Ltd: Nature, copyright 2011.

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Antifreeze Proteins. Spatially extended ordering of bound wateris observed in antifreeze proteins, which bind to ice to controlcrystallite nucleation and growth. For example, the arrangementof hydrogen-bonding moieties on the protein surface may becommensurate with those in the ice lattice, as seen, for example,in wfAFP-1 (71). The involvement of bound water can be moreexotic. The fish antifreeze protein Maxi is a four-helix bundle withan interior, mostly hydrophobic channel filled with more than400 water molecules, crystallographically ordered into a clathrate-like network of predominantly five-membered rings (72). It seemsthat this ordered network extends outward through the gapsbetween the helices to create an ordered layer of water moleculeson the outer surface that enables Maxi to bind to ice crystals and

hinder their growth, acting as a kind of “molecular Velcro” for icebinding (Fig. 5).

Water in Ligand Binding and Drug DesignThe structural participation of hydration water in biomolecularrecognition recommends it as a potential element in drug design.The difficulty, however, is that it is far from obvious how to gener-alize such behavior to extract design rules. Until recently, therefore,there has been relatively little effort to make use of the versatility ofhydration water in this manner (73–76).

In general, water networks are rearranged and/or displaced byligand binding—and here, the thermodynamic consequencesmay be subtle and hostage to fine details. That difficulty was

Fig. 5. (A) The network of some 400 interior waters in the antifreeze protein Maxi, with hydrogen bonds indicated by dotted lines. (B) Cross-sectionshowing part of the water network rich in five-membered clathrate-like rings. Reproduced from ref. 72 with permission from AAAS.

Fig. 4. Water-assisted excision of a damaged adenine in DNA byMutY. (Left) The mispaired adenine is extruded into a cavity. (Right) The catalyticsite contains five water molecules. Proton transfer from E43 to N7 is mediated by W1, supported by neighboring structured water molecules.Reproduced from ref. 66 with permission, copyright 2012 American Chemical Society.

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made particularly clear in studies by Whitesides and coworkers(77–79) of protein–ligand binding in the human carbonic anhy-drase system. A series of ligands modified with various thiazole-based sulfonamides was used to probe the effects of solventrearrangements within the binding cavity. The changes in the freeenergy of binding and the contributions from enthalpy and en-tropy are largely determined by the rearrangements or displace-ments of water: Breiten et al. (78) conclude that a “water-centric”view of ligand binding cannot be rationalized by the lock and keyprinciple; rather, the molecules of water surrounding the ligandand filling the active site of a protein are as important as thestructure of the ligand and the surface of the active site. However,there is nothing obvious about how water molecules function inthis capacity. Although it is tempting, for example, to imagine thatbinding will be governed by the entropic benefit of expellingbound water from the hydrophobic face of the binding pocket, infact enthalpic effects at the hydrophilic surface seem to pre-dominate in this case.

Despite such complexities, rational design of ligands anddrugs that takes water-mediated interactions into account doesnow seem to be becoming feasible. Consider, for instance, theM2 proton channel of the influenza A virus, a common drug tar-get. Here, a water network seems to gate proton conduction (58,80). Gianti et al. (81) have studied how the proton channel may betargeted with inhibitory drugs. Known inhibitors seem to bind tothe channel and disrupt the proton-transporting water cluster. Bycalculating the energetics of pore blockers at different sites in M2,they conclude that effective ligand scaffolds mimic the watercluster contour, while also preserving the interactions that thecluster made with the protein.

Krimmer et al. (82) focus instead on the nature of hydration ofthe final protein–ligand complex. They say that optimizing thewater layers covering hydrophobic inhibitors of thermolysin canboost the enthalpic contribution to binding free energy. Theirsimulations enabled the prediction of high binding affinity for aseries of ligands, one of which was subsequently shown experi-mentally to have 50 times higher binding affinity than the known,patented parent ligand.

Perhaps data-mining and empirical correlations will, in the end,offer more in the way of rules of thumb for ligand design than willstudies from first principles. By analyzing over 2,000 crystalstructures of hydrated and nonhydrated ligand–receptor com-plexes, including many drugs, Garcıa-Sosa (83) finds, for example,that bridging water molecules are effective targets for achievingtight binding.

There is increasing reason to believe that the efficiency of li-gand binding is also influenced by dynamical aspects of hydra-tion. Studies using terahertz spectroscopy have shown thatmodified dynamics can appear in a protein’s hydration shell up toat least 10 Å from the protein surface (84). On these timescales,the solvent motions involve collective modes of many watermolecules. The metalloprotease MT1-MMP establishes a dy-namical gradient close to the active site as the substrate ap-proaches, creating a “hydration funnel” that guides the molecularrecognition process by reducing the entropic cost of binding (84).Long-range control of water dynamics, extending up to 20 Å fromthe molecular surface, also seems important for the ice bindingfunction of some antifreeze proteins and glycoproteins (85, 86).

ConclusionInsights gleaned over the past two decades or so about the rolesof water in molecular and cell biology leave no doubt that it exerts

an active agency in life, extending, modifying, complementing,and enabling the functions of biomolecules. Many questions re-main open. How are the dynamics of water and biomolecularsolutes related, and how do these dynamics influence function?How are fluctuations on different timescales and spatial scalescoupled? How are the properties of water modulated at surfaces,and how do these depend on the chemical and geometric fea-tures of the surface? How are these properties modified by thepresence of cosolutes, such as salts and small osmolyte mole-cules? How does hydration affect the behaviors of membranes,nucleic acids, and glycoproteins, which have been afforded ratherless attention than proteins? Does hydration play a role in disease(for example, in mediating protein misfolding)? Are there gen-eral principles of hydration that can be exploited in ligand anddrug design?

Broader questions on which it is, at this stage, perhaps possi-ble to do no more than speculate are whether, where, and by howmuch the biomolecular uses of hydration in function are evolu-tionarily adaptive. It could be that—like, for example, examples ofquantum mechanical tunneling in biomolecular proton and elec-tron transfer—at least some of these manifestations are an in-evitable physicochemical outcome rather than an adaptation.However, whatever the case, the sensitivity of the hydration en-vironment, exploiting the cooperativity of water motions, to smallchanges in protein conformation offers a sensitive way for mac-romolecular dynamics to alter behavior. As De Simone et al. (87)have put it: “The sensitivity of the energy surfaces of proteins tominor perturbations supports the view that there is a delicatebalance between functionality, stability, and solubility, which isencapsulated by the concept of ‘life on the edge.’”

All of this touches on the astrobiological question mentionedat the outset: should we, like Henderson (88) in 1913, regardwater as uniquely “biophilic” and “fit” to act as life’s matrix? Theanswer remains unclear, although, given that there are severalpotential alternative astrobiological solvents (6), we would bewell-advised not to take it for granted. However, what the currentunderstanding of biological hydration does tell us is that the is-sues are more subtle than is often supposed, because water’sbiological roles are not easily reduced to a handful of propertiesthat follow self-evidently from its molecular nature, or indeedeven from the characteristics of the pure bulk liquid. We mightsay, however, that water does seem rather special in offering adegree of what one may call biological affordance: it offers op-portunities for refining and conditioning intermolecular in-formation transfer that seem less readily available from othersolvents. [I use “affordance” here in Gibson’s (89) original senseof opportunities provided by the environment.]

It can be hard to keep an appropriate perspective when wateris concerned. As a substance of primal significance to humanculture, it has a propensity to generate mythology that becomesmanifest even at the scale of basic physics and chemistry. Its no-torious roles in episodes of pathological science, such as poly-water and “water memory,” are the most egregious instances, butthere is a whiff of this mythopoeic character in discussions of“vicinal water” (90), biological water, and water structure invokedto explain, inter alia, long-ranged hydrophobic (91) and hydro-philic (92) interactions and Hofmeister or ion-specific effects ofelectrolytes on protein aggregation (93). We should approachwater’s biological behavior with this in mind, wary not to claim toomuch on its behalf. To take just one example, the alluring conceptof solvent “structure making and breaking” as an explanatoryscheme for the biological effects of ions, denaturants, and other

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osmolytes seems now to be a simplistic residue of attempts to use“water structure” to explain puzzling solvation phenomena. Suchcosolute effects are better understood by delving into the specificdetails of direct interactions between solutes and biomolecules(94). This is not to suggest that water structure is in itself an ob-solete concept: there seems no doubt that hydration must takeaccount of structural aspects, such as tetrahedrality, coordinationnumber, and dangling bonds. The point is that, first, it is hardto generalize about such things, especially when discussing

biomolecular hydration, and second, dynamical factors oftenseem to be at least as important as any static structural pictures.

However, if we should abandon notions of some special andwell-defined phase called biological water, there does not seemto be any prospect of or virtue in returning water to its humbleposition of life’s canvas. It is a versatile, responsive medium thatblurs the boundaries between mechanism and matrix. It surely isspecial; we might have to depend on either synthetic biology orobservational astrobiology to tell us just how special.

1 Ojha L, et al. (2015) Spectral evidence for hydrated salts in recurring slope lineae on Mars. Nat Geosci 8:829–832.2 Chaplin M (2006) Do we underestimate the importance of water in cell biology? Nat Rev Mol Cell Biol 7:861–866.3 Ball P (2008) Water as an active constituent in cell biology. Chem Rev 108:74–108.4 Franks F, ed (1972) Water: A Comprehensive Treatise (Plenum, New York), Vol 1.5 Stanley HE, et al. (1999) The puzzle of water: A very complex fluid. Physica D 133:453–462.6 Benner SA, Ricardo A, Carrigan MA (2004) Is there a common chemical model for life in the universe? Curr Opin Chem Biol 8:672–689.7 Sterpone F, Derreumaux P, Melchionna S (2015) Protein simulations in fluids: Coupling the OPEP coarse-grained force field with hydrodynamics. J Chem TheoryComput 11:1843–1853.

8 Mentre P (2001) An introduction to “Water in the cell”: Tamed Hydra? Cell Mol Biol 47:709–715.9 Jungwirth P (2015) Biological water or rather water in biology? J Phys Chem Lett 6:2449–2451.

10 Persson E, Halle B (2008) Cell water dynamics on multiple time scales. Proc Natl Acad Sci USA 105:6266–6271.11 Jasnin M, Moulin M, Haertlein M, Zaccai G, Tehei M (2008) Down to atomic-scale intracellular water dynamics. EMBO Rep 9:543–547.12 Ball P (2014) The hidden structure of liquids. Nat Mater 13:758–759.13 Stillinger FH (1980) Water revisited. Science 209:451–457.14 Soper AK, Castner EW, Luzar A (2003) Impact of urea on water structure: A clue to its properties as a denaturant? Biophys Chem 105:649–666.15 Finney JL (2004) Water? What’s so special about it? Philos Trans R Soc Lond B Biol Sci 359:1145–1163.16 Cappa CD, Smith JD, Messer BM, Cohen RC, Saykally RJ (2006) Effects of cations on the hydrogen bond network of liquid water: New results from X-ray

absorption spectroscopy of liquid microjets. J Phys Chem B 110:5301–5309.17 Kauzmann W (1959) Some factors in the interpretation of protein denaturation. Adv Protein Chem 14:1–63.18 Tanford C (1980) The Hydrophobic Effect (Wiley, New York), 2nd Ed.19 Blokzijl W, Engberts JBFN (1993) Hydrophobic effects. Opinions and facts. Angew Chem Int Ed Engl 32:1545–1579.20 Duboue-Dijon E, Laage D (2015) Characterization of the local structure in liquid water by various order parameters. J Phys Chem B 119:8406–8418.21 Kirchner B, Stubbs J, Marx D (2002) Fast anomalous diffusion of small hydrophobic species in water. Phys Rev Lett 89:215901.22 Baldwin RL (2014) Dynamic hydration shell restores Kauzmann’s 1959 explanation of how the hydrophobic factor drives protein folding. Proc Natl Acad Sci USA

111:13052–13056.23 Lum K, Chandler D, Weeks JD (1999) Hydrophobicity at small and large length scales. J Phys Chem B 103:4570–4577.24 Chandler D (2005) Interfaces and the driving force of hydrophobic assembly. Nature 437:640–647.25 Montagna M, Sterpone F, Guidoni L (2012) Structural and spectroscopic properties of water around small hydrophobic solutes. J Phys Chem B 116:11695–11700.26 Rezus YLA, Bakker HJ (2007) Observation of immobilized water molecules around hydrophobic groups. Phys Rev Lett 99:148301.27 Qvist J, Halle B (2008) Thermal signature of hydrophobic hydration dynamics. J Am Chem Soc 130:10345–10353.28 Titantah JT, Karttunen M (2012) Long-time correlations and hydrophobe-modified hydrogen-bonding dynamics in hydrophobic hydration. J Am Chem Soc

134:9362–9368.29 Vaikuntanathan S, Geissler PL (2014) Putting water on a lattice: The importance of long wavelength density fluctuations in theories of hydrophobic and interfacial

phenomena. Phys Rev Lett 112:020603.30 Remsing RC, et al. (2015) Pathways to dewetting in hydrophobic confinement. Proc Natl Acad Sci USA 112:8181–8186.31 Wallqvist A, Berne BJ (1995) Computer simulation of hydrophobic hydration forces on stacked plates at short range. J Phys Chem 99:2893–2899.32 Hua L, Huang X, Liu P, Zhou R, Berne BJ (2007) Nanoscale dewetting transition in protein complex folding. J Phys Chem B 111:9069–9077.33 Giovambattista N, Lopez CF, Rossky PJ, Debenedetti PG (2008) Hydrophobicity of protein surfaces: Separating geometry from chemistry. Proc Natl Acad Sci USA

105:2274–2279.34 Camilloni C, et al. (2016) Towards a structural biology of the hydrophobic effect in protein folding. Sci Rep 6:28285.35 Cheng Y-K, Rossky PJ (1998) Surface topography dependence of biomolecular hydrophobic hydration. Nature 392:696–699.36 Patel AJ, et al. (2012) Sitting at the edge: How biomolecules use hydrophobicity to tune their interactions and function. J Phys Chem B 116:2498–2503.37 Yu N, Hagan MF (2012) Simulations of HIV capsid protein dimerization reveal the effect of chemistry and topography on the mechanism of hydrophobic protein

association. Biophys J 103:1363–1369.38 Beckstein O, Biggin PC, Sansom MSPA (2001) A hydrophobic gating mechanism for nanopores. J Phys Chem B 105:12902–12905.39 Anishkin A, Sukharev S (2004) Water dynamics and dewetting transitions in the small mechanosensitive channel MscS. Biophys J 86:2883–2895.40 Yin H, Hummer G, Rasaiah JC (2007) Metastable water clusters in the nonpolar cavities of the thermostable protein tetrabrachion. J AmChem Soc 129:7369–7377.41 Godec A, Smith JC, Merzel F (2013) Soft collective fluctuations governing hydrophobic association. Phys Rev Lett 111:127801.42 Remsing RC, Weeks JD (2013) Dissecting hydrophobic hydration and association. J Phys Chem B 117:15479–15491.43 Cerdeiri~na CA, Debenedetti PG, Rossky PJ, Giovambattista N (2011) Evaporation length scales of confined water and some common organic liquids. J Phys Chem

Lett 2:1000–1003.44 Chacko B, Evans R, Archer AJ (2017) Solvent fluctuations around solvophobic, solvophilic, and patchy nanostructures and the accompanying solvent mediated

interactions. J Chem Phys 146:124703.45 Conti Nibali V, Havenith M (2014) New insights into the role of water in biological function: Studying solvated biomolecules using terahertz absorption

spectroscopy in conjunction with molecular dynamics simulations. J Am Chem Soc 136:12800–12807.46 Xu Y, Havenith M (2015) Perspective: Watching low-frequency vibrations of water in biomolecular recognition by THz spectroscopy. J Chem Phys 143:170901.47 Mondal S, Mukherjee S, Bagchi B (2017) Dynamical coupling between protein conformational fluctuation and hydration water: Heterogeneous dynamics of

biological water. arXiv:1701.04861.48 Makarov VA, Andrews BK, Smith PE, Pettitt BM (2000) Residence times of water molecules in the hydration sites of myoglobin. Biophys J 79:2966–2974.49 Persson F, Halle B (2013) Transient access to the protein interior: Simulation versus NMR. J Am Chem Soc 135:8735–8748.

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Dow

nloa

ded

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50 Brotzakis ZF, Groot CCM, Brandeburgo WH, Bakker HJ, Bolhuis PG (2016) Dynamics of hydration water around native and misfolded α-lactalbumin. J Phys ChemB 120:4756–4766.

51 Fogarty AC, Laage D (2014) Water dynamics in protein hydration shells: The molecular origins of the dynamical perturbation. J Phys Chem B 118:7715–7729.52 Frauenfelder H, Sligar SG, Wolynes PG (1991) The energy landscapes and motions of proteins. Science 254:1598–1603.53 Wang Z, et al. (2013) Inelastic X-ray scattering studies of the short-time collective vibrational motions in hydrated lysozyme powders and their possible relation to

enzymatic function. J Phys Chem B 117:1186–1195.54 Frauenfelder H, et al. (2009) A unified model of protein dynamics. Proc Natl Acad Sci USA 106:5129–5134.55 Ball P (2008) Water as a biomolecule. ChemPhysChem 9:2677–2685.56 Agmon N (1995) The Grotthuss mechanism. Chem Phys Lett 244:456–462.57 Hassanali A, Giberti F, Cuny J, Kühne TD, Parrinello M (2013) Proton transfer through the water gossamer. Proc Natl Acad Sci USA 110:13723–13728.58 Swanson JMJ, et al. (2007) Proton solvation and transport in aqueous and biomolecular systems: Insights from computer simulations. J Phys Chem B

111:4300–4314.59 Kaila VRI, WikströmM, Hummer G (2014) Electrostatics, hydration, and proton transfer dynamics in the membrane domain of respiratory complex I. Proc Natl Acad

Sci USA 111:6988–6993.60 Goyal P, Lu J, Yang S, Gunner MR, Cui Q (2013) Changing hydration level in an internal cavity modulates the proton affinity of a key glutamate in cytochrome c

oxidase. Proc Natl Acad Sci USA 110:18886–18891.61 Lorch S, Capponi S, Pieront F, Bondar A-N (2015) Dynamic carboxylate/water networks on the surface of the PsbO subunit of photosystem II. J Phys Chem B

119:12172–12181.62 Umena Y, Kawakami K, Shen J-R, Kamiya N (2011) Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å. Nature 473:55–60.63 Polander BC, Barry BA (2012) A hydrogen-bonding network plays a catalytic role in photosynthetic oxygen evolution. Proc Natl Acad Sci USA 109:6112–6117.64 Polander BC, Barry BA (2013) Detection of an intermediary, protonated water cluster in photosynthetic oxygen evolution. Proc Natl Acad Sci USA

110:10634–10639.65 Brahmachari U, Barry BA (2016) Dynamics of proton transfer to internal water during the photosynthetic oxygen-evolving cycle. J Phys Chem B 120:11464–11473.66 Brunk E, Arey JS, Rothlisberger U (2012) Role of environment for catalysis of the DNA repair enzyme MutY. J Am Chem Soc 134:8608–8616.67 Oroguchi T, Nakasako M (2016) Changes in hydration structure are necessary for collective motions of a multi-domain protein. Sci Rep 6:26302.68 Buchli B, et al. (2013) Kinetic response of a photoperturbed allosteric protein. Proc Natl Acad Sci USA 110:11725–11730.69 Hou G, Cui Q (2013) Stabilization of different types of transition states in a single enzyme active site: QM/MM analysis of enzymes in the alkaline phosphatase

superfamily. J Am Chem Soc 135:10457–10469.70 Pal R, Sekharan S, Batista VS (2013) Spectral tuning in halorhodopsin: The chloride pump photoreceptor. J Am Chem Soc 135:9624–9627.71 Ebbinghaus S, et al. (2012) Functional importance of short-range binding and long-range solvent interactions in helical antifreeze peptides. Biophys J

103:L20–L22.72 Sun T, Lin FH, Campbell RL, Allingham JS, Davies PL (2014) An antifreeze protein folds with an interior network of more than 400 semi-clathrate waters. Science

343:795–798.73 Renzoni DA, Zvelebil MJJM, Lundbäck T, Ladbury JE (1997) Exploring uncharted waters: Water molecules in drug design strategies. Structure-Based Drug Design:

Thermodynamics, Modeling and Strategy, eds Ladbury JE, Connelly PR (Landes Bioscience, Austin, TX), pp 161–180.74 Ladbury JE (1996) Just add water! The effect of water on the specificity of protein-ligand binding sites and its potential application to drug design. Chem Biol

3:973–980.75 Henriques DA, Ladbury JE (2001) Inhibitors to the Src SH2 domain: A lesson in structure–thermodynamic correlation in drug design. Arch Biochem Biophys

390:158–168.76 Barillari C, Taylor J, Viner R, Essex JW (2007) Classification of water molecules in protein binding sites. J Am Chem Soc 129:2577–2587.77 Snyder PW, et al. (2011) Mechanism of the hydrophobic effect in the biomolecular recognition of arylsulfonamides by carbonic anhydrase. Proc Natl Acad Sci USA

108:17889–17894.78 Breiten B, et al. (2013) Water networks contribute to enthalpy/entropy compensation in protein-ligand binding. J Am Chem Soc 135:15579–15584.79 Snyder PW, Lockett MR, Moustakas DT, Whitesides GM (2014) Is it the shape of the cavity, or the shape of the water in the cavity? Eur Phys J Spec Top

223:853–891.80 Wu Y, Voth GA (2005) A computational study of the closed and open states of the influenza a M2 proton channel. Biophys J 89:2402–2411.81 Gianti E, Carnevale V, DeGrado WF, Klein ML, Fiorin G (2015) Hydrogen-bonded water molecules in the M2 channel of the influenza A virus guide the binding

preferences of ammonium-based inhibitors. J Phys Chem B 119:1173–1183.82 Krimmer SG, et al. (2016) Rational design of thermodynamic and kinetic binding profiles by optimizing surface water networks coating protein-bound ligands.

J Med Chem 59:10530–10548.83 Garcıa-Sosa AT (2013) Hydration properties of ligands and drugs in protein binding sites: Tightly-bound, bridging water molecules and their effects and

consequences on molecular design strategies. J Chem Inf Model 53:1388–1405.84 Grossman M, et al. (2011) Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site. Nat Struct Mol Biol 18:1102–1108.85 Ebbinghaus S, et al. (2010) Antifreeze glycoprotein activity correlates with long-range protein-water dynamics. J Am Chem Soc 132:12210–12211.86 Meister K, et al. (2013) Long-range protein-water dynamics in hyperactive insect antifreeze proteins. Proc Natl Acad Sci USA 110:1617–1622.87 De Simone A, et al. (2011) Experimental free energy surfaces reveal the mechanisms of maintenance of protein solubility. Proc Natl Acad Sci USA

108:21057–21062.88 Henderson L (1913) The Fitness of the Environment (Macmillan, New York).89 Gibson JJ (1977) The theory of affordances. Perceiving, Acting, and Knowing, eds Shaw R, Bransford J (Erlbaum, Hillsdale, NJ), pp 127–143.90 Drost-Hansen W (1978) Water at biological interfaces: Structural and functional aspects. Phys Chem Liquids 7:243–348.91 Pashley RM,McGuiggan PM, Ninham BW, Evans DF (1985) Attractive forces between uncharged hydrophobic surfaces: Direct measurements in aqueous solution.

Science 229:1088–1089.92 Israelachvili J, Wennerström H (1996) Role of hydration and water structure in biological and colloidal interactions. Nature 379:219–225.93 Marcus Y (2009) Effect of ions on the structure of water: Structure making and breaking. Chem Rev 109:1346–1370.94 Ball P, Hallsworth JE (2015) Water structure and chaotropicity: Their uses, abuses and biological implications. Phys Chem Chem Phys 17:8297–8305.

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